In vivo behavior of muscle fascicles and tendinous tissues in human tibialis anterior muscle during twitch contraction

In this study we investigated the time course of length and velocity of muscle fascicles and tendinous tissues (TT) during isometric twitch contraction, and examined how their interaction relates to the time course of external torque and muscle fascicle force generation. From seven males, supra-maximal twitch contractions (singlet) of the tibialis anterior muscle were induced at 30 degrees , 10 degrees and -10 degrees plantar flexed positions. The length and velocity of fascicles and TT were determined from a series of their transverse ultrasound images. The maximal external torque appeared when the shortening velocity of fascicles was zero. The fascicle and TT length, and external torque showed a 10-30 ms delay of each onset, with a significant difference in half relaxation times at -10 degrees . The time course of TT elongation, and fascicle and tendinous velocities did not differ between joint angles. Curvilinear length-force properties, whose slope of quasi-linear part was ranged from -15.0 to -5.9 N/mm for fascicles and 5.4 to 14.3N/mm for TT, and a loop-like pattern of velocity-force properties, in which the mean power was ranged from 0.14 to 0.80 W for fascicles, and 0.14 to 0.81 W for TT were also observed. These results were attributed to the muscle-tendon interaction, depending on the slack and non-linearity of length-force relationship of compliant TT. We conclude that the mechanical interaction between fascicles and TT, are significant determinants of twitch force and time characteristics.     

Changes of cerebral blood oxygenation and optical pathlength during activation and deactivation in the prefrontal cortex measured by time-resolved near infrared spectroscopy

To determine the alterations in optical characteristics and cerebral blood oxygenation (CBO) during activation and deactivation, we evaluated the changes in mean optical pathlength (MOP) and CBO induced by a verbal fluency task (VFT) and driving simulation in the right and left prefrontal cortex (PFC), employing a newly developed time-resolved near infrared spectroscopy, which allows quantitative measurements of the evoked-CBO changes by determining the MOP with a sampling time of 1 s. The results demonstrated differences in MOP in the foreheads with the subjects and wavelength; however, there was no significant difference between the right and left foreheads (p > 0.05). Also, both the VFT and driving simulation task did not affect the MOP significantly as compared to that before the tasks (p > 0.05). In the bilateral PFCs, the VFT caused increases of oxyhemoglobin and total hemoglobin associated with a decrease of deoxyhemoglobin, while the driving simulation task caused decreases of oxyhemoglobin and total hemoglobin associated with an increase of deoxyhemoglobin; there were no significant differences in evoked-CBO changes between the right and left PFC. The present results will be useful for quantitative measurement of hemodynamic changes during activation and deactivation in the adults by near infrared spectroscopy.     

Insulin mimetic effect of a tungstate cluster. Effect of oral administration of homo-polyoxotungstates and vanadium-substituted polyoxotungstates on blood glucose level of STZ mice

Aqueous vanadate and aqueous tungstate have been known to mimic all or most of the actions of insulin in intact cell systems with respect to normalization of the blood glucose level. By carrying out oral administration in vivo experiments on the blood glucose level of streptozotocin (STZ)-induced diabetes (STZ mice), the insulin-mimetic (IM) effects of metal-oxide clusters of all-inorganic composition were examined using many types of polyoxometalates (POM) with and without vanadium substitution. Several homo-POM and vanadium-substituted POM showed hypoglycemic effects. The observed hypoglycemic effects indicated that POM with the Dawson structure [[alpha-P(2)W(18)O(62)](6-) (W-2), [alpha-P(2)W(17)V(V)O(62)](7-) (V-19) and [alpha-1,2,3-P(2)W(15)V(V)(3)O(62)](9-) (V-04)] are more effective than those with the Keggin structure [[alpha-PW(12)O(40)](3-) (W-1), [alpha-PW(11)V(V)O(40)](4-) (V-01), [alpha-1,2-PW(10)V(V)(2)O(40)](5-) (V-02), [alpha-1,2,3-PW(9)V(V)(3)O(40)](6-) (V-03) and [alpha-1,4,9-PW(9)V(V)(3)O(40)](6-) (V-13)]. The vanadate cluster [V(10)O(28)](6-) (V-15) also showed a hypoglycemic effect. (31)P and (51)V NMR measurements showed that the Dawson POM (W-2, V-04 and V-19) are stable in aqueous solution under the conditions used. The effect of all POM on the body weight of STZ mice was also examined. The decrease in body weight after administration of W-2 was much less than for V-19, V-04 and V-15. This suggests that not only monomeric tungstate and vanadate, but also the structure factors of tungstate and vanadate clusters, can play a significant role in their biological action.     

Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.     

Effects of intranasal administration of atrial natriuretic hormone on spontaneously hypertensive rats

The effects of the intranasal administration of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) were investigated in 14 anesthetized spontaneously hypertensive rats (SHR; Okamoto-Aoki strain). They were given intranasally synthetic alpha-hANP in distilled water at doses of 10 micrograms/kg, 50 micrograms/kg and 100 micrograms/kg. Intranasal application of 200 microliter of distilled water as a control was also performed in 3 anesthetized SHR. Sixteen anesthetized SHR were examined for the effects of intravenous administration of alpha-hANP at doses of 4 micrograms/kg, 10 micrograms/kg, 20 micrograms/kg and 40 micrograms/kg. Urinary volume and the urinary excretion of sodium increased 2- to 3-fold during the 50 minutes following intranasal administration of a single dose of 50 micrograms/kg or 100 micrograms/kg, although neither the urinary volume nor the urinary excretion of sodium increased after intranasal administration of 10 micrograms/kg of alpha-hANP or 200 microliter of distilled water. There were no significant changes in arterial pressure or heart rate after the intranasal administration of synthetic alpha-hANP or distilled water. In contrast, arterial pressure was decreased and urinary volume and urinary excretion of sodium were increased, in a dose dependent manner, within 5 minutes after intravenous bolus-injection of alpha-hANP and returned to their baseline levels within 20 minutes. These results indicate that intranasal administration of synthetic alpha-hANP exerts its diuretic effect without concomitant changes in arterial pressure or heart rate in SHR.     

Primary structure,unique enzymatic properties,and molecular evolution of pepsinogen B and pepsin B

Purification of pepsinogen B from dog stomach was achieved. Activation of pepsinogen B to pepsin B is likely to proceed through a one-step pathway although the rate is very slow. Pepsin B hydrolyzes various peptides including beta-endorphin, insulin B chain, dynorphin A, and neurokinin A, with high specificity for the cleavage of the Phe-X bonds. The stability of pepsin B in alkaline pH is noteworthy, presumably due to its less acidic character. The complete primary structure of pepsinogen B was clarified for the first time through the molecular cloning of the respective cDNA. Molecular evolutional analyses show that pepsinogen B is not included in other known pepsinogen groups and constitutes an independent cluster in the consensus tree. Pepsinogen B might be a sister group of pepsinogen C and the divergence of these two zymogens seems to be the latest event of pepsinogen evolution.     

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.     

Capture,immobilization, and Global Positioning System collaring of olive baboons (Papio anubis) and vervets (Chlorocebus pygerythrus): Lessons learned and suggested best practices

As the value of Global Positioning System (GPS) technology in addressing primatological questions becomes more obvious, more studies will include capturing and collaring primates, with concomitant increased risk of adverse consequences to primate subjects. Here we detail our experiences in capturing, immobilizing, and placing GPS collars on six olive baboons (Papio anubis) in four groups and 12 vervet monkeys (Chlorocebus pygerythrus) in five groups in Kenya. We captured baboons with cage traps and vervets with box traps, immobilized them, and attached GPS collars that were to be worn for 1 year. Adverse consequences from the trapping effort included incidental death of two nonsubjects (an adult female and her dependent infant), temporary rectal prolapse in one baboon, superficial wounds on the crown of the head in two vervets, and failure to recapture/remove collars from two baboons and two vervets. Obvious negative effects from wearing collars were limited to abrasions around the neck of one vervet. A possible, and if so, serious, adverse effect was greater mortality for collared adult female vervets compared with known uncollared adult female vervets, largely due to leopard (Panthera pardus) predation. Collared animals could be more vulnerable to predation because trapping favors bolder individuals, who may also be more vulnerable to predation, or because collars could slow them down or make them more noticeable to predators. Along with recommendations made by others, we suggest that future studies diversify trapping bait to minimize the risk of rectal prolapse, avoid capturing the first individuals to enter traps, test the movement speeds of collared versus noncollared animals, include a release system on the collars to avoid retrapping failure, and publish both positive and negative effects of capturing, immobilizing, and collaring.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.     

Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes.

The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.